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Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Agonist-selective patterns of µ-opioid receptor phosphorylation revealed by phosphosite-specific antibodies

Christian Doll1, Jens Konietzko, Florian Pöll

  • 1Institute of Pharmacology and Toxicology, University Hospital, Friedrich Schiller University, Jena, Germany.

British Journal of Pharmacology
|April 1, 2011
PubMed
Abstract

Insights

Morphine and full opioid agonists trigger distinct µ-opioid receptor phosphorylation patterns. These site-specific phosphorylations correlate with the receptor

Area of Science:

  • Pharmacology
  • Molecular Biology
  • Cellular Signaling

Background:

  • Morphine activates the µ-opioid receptor (MOR) without rapid endocytosis.
  • Full MOR agonists like DAMGO and etonitazene induce rapid and profound receptor internalization.
  • The molecular mechanisms behind differential MOR trafficking regulation by agonists are not fully understood.

Purpose of the Study:

  • To investigate the site-specific phosphorylation patterns of the MOR induced by different agonists.
  • To elucidate the relationship between MOR phosphorylation and receptor trafficking (sequestration).

Main Methods:

  • Generation of phosphosite-specific antibodies for MOR carboxyl-terminal residues Ser363, Thr370, and Ser375.
  • Detection of phosphorylated MOR forms using these novel antibodies.
  • Analysis of agonist-induced and PKC-mediated phosphorylation events.

Main Results:

  • Agonist-induced MOR phosphorylation occurs at Thr370 and Ser375; Ser363 is constitutively phosphorylated.
  • DAMGO and etonitazene stimulate phosphorylation at both Thr370 and Ser375.
  • Morphine stimulates Ser375 phosphorylation but not Thr370 phosphorylation.
  • Ser375 phosphorylation is faster than Thr370 phosphorylation, indicating it's a primary site.
  • PKC activation phosphorylates Thr370 but not Ser375, suggesting heterologous phosphorylation.
  • Dephosphorylation occurs rapidly upon agonist removal, especially for Thr370 and Ser375.

Conclusions:

  • Distinct opioid agonists induce unique MOR phosphorylation profiles.
  • These agonist-specific phosphorylation patterns are directly linked to the receptor's sequestration.
  • This study provides novel insights into the molecular regulation of MOR trafficking.

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